The transforming growth factor-beta (TGF.beta.) family of structurally related paracrine polypeptides are found ubiquitously in vertebrates, and are prototypic of a large family of metazoan growth, differentiation, and morphogenesis factors (see, for review, Massaque et al. (1990) Ann Rev Cell Biol 6:597-641; Massaque et al. (1994) Trends Cell Biol. 4:172-178; Kingsley (1994) Gene Dev. 8:133-146; and Spom et al. (1992) J Cell Biol 119:1017-1021). As described in Kingsley, supra, the TGF.beta. superfamily has at least 25 members, and can be grouped into distinct sub-families with highly related sequences. The most obvious sub-families include the following: the TGF.beta. sub-family, which comprises at least four genes that are much more similar to TGF.beta.-1 than to other members of the TGF.beta. superfamily; the activin sub-family, comprising homo- or heterodimers or two subunits, inhibin.beta.-A and inhibin.beta.-B; the decapentaplegic (DPP) subfamily, which includes the mammalian factors BMP2 and BMP4, which can induce the formation of ectopic bone and cartilage when implanted under the skin or into muscles; and the 60A subfamily, which includes a number of mammalian homologs, with osteoinductive activity, including BMP5-8. Other members of the TGF.beta. superfamily include the gross differentiation factor 1(GDF-1), GDF-3/VGR-2, dorsalin, nodal, mullerian-inhibiting substance (MIS), and glial-derived neurotrophic growth factor (GDNF). The DPP and 60A subfamilies are related more closely to one another than to other members of the TGF.beta. superfamily, and have often been grouped together as part of a larger collection of molecules called DVR (dpp and vg1 related).
Signals transduced by the TGF.beta. superfamily of molecules play salient roles in numerous diverse biologic processes, including inflammation and host defense, in addition to development, tissue repair, and tumorigenesis (Wahl. 1994. J Exp. Med. 180:1587). Members of the family are initially synthesized as larger precursor molecules with an amino-terminal signal sequence and a pro-domain of varying size (Kingsley, D. M. (1994) Genes Dev. 8:133-146). The precursor is then cleaved to release a mature carboxy-terminal segment of 110-140 amino acids. The active signaling moiety is comprised of hetero- or homodimers of the carboxy-terminal segment (Massague, J. (1990) Annu. Rev. Cell Biol. 6:597-641). The active form of the molecule then interacts with its receptor, which for this family of molecules is composed of two distantly related transmembrane serine/threonine kinases called type I and type II receptors (Massague, J. et al. (1992) Cell 69:1067-1070; Miyazono, K. A. et al. EMBO J. 10:1091-1101). TGF.beta. binds directly to the type II receptor, which then recruits the type I receptor and modifies it by phosphorylation. The type I receptor then transduces the signal to downstream components, which are as yet unidentified (Wrana et al, (1994) Nature 370:341-347).
Several members of the TGF.beta. superfamily have been identified which play roles during mammalian development. For example, dorsalin is expressed preferentially in the dorsal side of the developing chick neural tube (Basler et al. (1993)Cell 73:687-702). It promotes the outgrowth of neural crest cells and inhibits the formation of motor neuron cells in vitro, suggesting that it plays an important role in neural patterning along the dorsoventral axis. Certain of the bone morphogenetic proteins (BMPs) can induce the formation of ectopic bone and cartilage when implanted under the skin or into muscles (Wozney, J. M. et al. (1988) Science 242:1528-1534). Members of the activin subfamily have been found to be important in mesoderm induction during Xenopus development (Green and Smith (1990) Nature 47:391-394; Thomsen et al. (1990) Cell 63:485-493) and inhibins were initially described as gonadal inhibitors of follicle-stimulating hormone from pituitary cells. In addition, antagonists of this signaling pathway can be used to convert embryonic tissue into ectoderm, the default pathway of development in the absence of TGF.beta.-mediated signals. BMP-4 and activin have been found to be potent inhibitors of neuralization (Wilson, P. A. and Hemmati-Brivanlou, A (1995) Nature 376:331-333).
Further evidence for the importance of a TGF.beta. family member in early mammalian development comes from a retroviral insertion in the mouse nodal gene. This insertion leads to a failure to form the primitive streak in early embryogenesis, a lack of axial mesoderm tissue, and an overproduction of ectoderm and extraembryonic ectoderm (Conlon et al. (1991) Development 111:969-981; Iannaccone et al (1992) Dev. Dynamics 194:198-208). The predicted nodal gene product is consistent with previous studies showing that nodal is related to activins and BMPs (Zhou et al. (1993) Nature 361:543-547). A role for TGF.beta. family members in the development of sex organs has also been described; Mullerian inhibitory substance functions during mammalian male sexual development to cause regression of the embryonic duct system that develops into oviducts and uterus (Lee and Donahoe (1993) Endocrinol. Rev. 14:152-164).
Members of this family of signaling molecules also continue to function post-development. TGF.beta. has a variety of pleiotropic effects on the immune system; it is capable of both stimulating inflammation, and immunosuppression. The apparent contradictory role of TGF.beta. in the immune system may, in part, be accounted for by differential effects of TGF.beta. on resting and activated cells (Wahl, supra.). Animals which cannot produce TGF.beta.1 (homozygous for null mutations in the TGF.beta.1 gene) have been found to survive until birth with no apparent morphological abnormalities (Shull et al. (1992) Nature 359:693-699; Kulkarni et al. (1993) Proc. Natl. Acac. Sci. 90:770-774). The animals do die around weaning age, however, owing to massive immune infiltration in many different organs, consistent with the inhibitory effects of TGF.beta. on lymphocyte growth (Tada et al. (1991) J. Immunol 146:1077-1082). The administration of TGF.beta. has been found to ameliorate experimental autoimmune encephalomyelitis and experimentally induced arthritis, thus demonstrating that modulation of TGF.beta. is a viable treatment for certain human autoimmune diseases (Border and Nobel, 1995 Nature Medicine 1:1000).
TGF.beta. can also promote neovascularization and the proliferation of connective tissue cells. Because of these activities, it plays a key role in wound healing (Kovacs, E. J. (1991) Immunol Today 12:17-23). Under normal conditions, TGF.beta. maintains cell numbers in the extracellular matrix by directly inhibiting cell proliferation and controlling the activity of platelet derived growth factor (Border and Nobel. supra.). Misexpression of TGF.beta. has been implicated in several disease processes, such as impaired wound healing in the elderly or diabetics. On the other hand, overproduction of TGF.beta. leads to the accumulation of pathological amounts of extracellular matrix, and may mediate fibrotic disorders affecting kidney, liver, lung, bone marrow, heart and skin (Border and Noble. 1994. New Engl. J. Med. 331:1286). Therefore, modulation of TGF.beta. may be useful in the treatment of disorders involving inappropriate healing and fibrosis.
Another proliferative disorder in which TGF.beta. has been implicated is the development of the atherosclerotic plaque. The advanced lesions of atherosclerosis result from an excessive inflammatory-fibroproliferative response to numerous different forms of insult. For example, shear stresses are thought to be responsible for the frequent occurrence of atherosclerotic plaques in regions of the circulatory system where turbulent blood flow occurs, such as branch points and irregular structures. Lipoprotein has been shown to inhibit TGF.beta. activation (Kojima et al. J. Cell Biol. 113:1439). The earliest lesion in atherosclerosis is the fatty streak, comprising lipid, macrophages, and T cells, which leads to smooth muscle cell proliferation and the development of more severe lesions (Ross. 1993 Nature 362:801). Transgenic mice which overexpress lipoprotein have been shown to have decreased levels of active TGF.beta. in serum and in the aortic wall (Grainger et al. 1995. Nature Med. 1:1067). In addition, treatment with tamoxifen, a known stimulator of TGF.beta., can suppress the development of the fatty streak lesion in mice (Grainger et al. supra). Finally, treatment with anti-TGF.beta. has blocked intimal hyperplasia and restenosis in a rat model (Nikol et al. 1992. J. Clin. Invest. 90:1582).
Certain members of the TGF.beta. family have potent antiproliferative effects. In certain cancer cells conversion to a tumorigenic phenotype has been shown to be accompanied by reduced response to the growth-inhibitory effects of TGF.beta. (Manning et al. 1991. Oncogene 6:1471). Moreover, abnormalities in TGF.beta. receptors have been identified in several human maliganacies (Markowitz et al. 1995. Science. 268:1336). Recently, a role for members of the TGF-.beta. superfamily has been postulated in tumor suppression (Hahn et al. 1996. Science 271:350). A gene, referred to as DPC4, has been found to be homozygously deleted in approximately 30% of pancreatic carcinomas tested. DPC4 was found to be homologous to the Drosophila melanogaster MAD gene and the sma-2, sma-3, and sma-4 genes of C. elegans (Hahn et al. supra), members of the DPP signaling subfamily of TGF.beta. molecules. Xenopus homologues of MAD have recently been cloned and have been shown to be important in the induction of mesoderm (Graff et al. 1996. Cell 85:479). MADs function downstream of TGF.beta. receptors and may function in the nucleus (Hoodless et al. 1996. Cell 85:489). Applicant has found MAD to be homologous to the fchd534 gene.